Our long-term goal is to elucidate the mechanisms by which the spliceosome catalyzes pre-mRNA splicing and in particular to define the structural and functional role of RNA and protein in catalysis. Catalysis of pre-mRNA splicing by the spliceosome is a defining feature of eukaryotes. In humans, catalysis is required to excise ten introns on average for nearly every gene, and catalysis at alternative sites expands the proteome and contributes to the complexity of higher eukaryotes. Indeed, mutations in at least 15% of human diseases result from errors in splicing. Despite this importance of pre-mRNA splicing to eukaryotic gene expression, for decades our understanding of splicing catalysis has languished, particularly in comparison to our understanding of catalysis at the two other major stages of gene expression - transcription and translation. We have not even known whether catalysis is mediated by the RNA or protein parts of the spliceosome. However, through our collaborative efforts, we have recently provided definitive evidence that the RNA parts catalyze splicing, positioning two divalent metal ions for interaction with the scissile phosphates. Strikingly, our work also establishes that the spliceosome utilizes a catalytic mechanism indistinguishable from the catalytic mechanism of self-splicing group II RNA introns, extending the parallels with the group II intron to the atomic level at the catalytic core and providing strog support for the hypothesis that these two enzymes share common evolutionary origins. Despite this advance and other achievements in the field, many fundamental questions remain. For example, how are the nucleophiles in either splicing system activated for catalysis, how does the architecture of the spliceosomal catalytic core position the reactive sites for catalysis, and what is the role of protein in mediating RNA-based catalysis? To answer these questions, we aim to test a metal cluster model for catalysis of nuclear pre-mRNA splicing, to define the architecture and dynamics of the catalytic core of the spliceosome, and to define the essential role of protein in assisting RNA-based splicing catalysis. We will accomplish these aims through a continuation of our unique and synergistic collaboration that allows for a combined approach of chemistry, biochemistry and molecular genetics. This work will transform our understanding of the roles of RNA and protein in spliceosomal catalysis, with significant implications for understanding the origins and evolution of the spliceosome.